Botulinum Neurotoxin (BoNT) is a category A select agent with extreme potency (lethal dose of ˜1 ng/kg in humans). Upon infection, BoNT impairs neuronal synaptic transmission in the peripheral nervous system, which may lead to paralysis and eventual respiratory failure. The toxin has seven serotypes (A-G), each consisting of a heavy chain (Hc, 100 kDa) and a light chain (Lc, 50 kDa). The heavy chain enables binding of the toxin to neuronal cell surface receptors and induces translocation of the light chain across the cell membrane. Once inside the cell, the enzymatic light chain cleaves a specific peptide sequence in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, thereby inhibiting acetylcholine release and impairing neuronal function. Serotype A (BoNT/A), the most common variant found in human cases, cleaves the synaptosome-associated protein of 25 kDa (SNAP-25) within the SNARE complex. Information about Botulinum Neurotoxin A may be found in Cai, S., Singh, B. R., Critical Reviews in Microbiology, 33 (2007): 109-125, which is hereby incorporated by reference for any purpose. Serotypes C and E (BoNT/C and BoNT/E) also cleave SNAP-25 at other sites. VAMP-2 is another SNARE protein, and is cleaved by BoNT/B, BoNT/D, BoNT/F and BoNT/G at different sites. Information about VAMP-2 may be found, for example, in Chen, et. al. “Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin,” Journal of Biological Chemistry, 283, no. 30 (2008): 21153-21159. Tetanus neurotoxin (TeNT) also cleaves VAMP-2 at the same site as BoNT/B. Syntaxin 1a is also a SNARE protein, and is cleaved by BoNT/C.
One prominent method for detecting BoNT infection is the mouse bioassay, as described for example in Lindstrom, M., Korkeala, H., Clinical Microbiology Reviews, 29 (2006): 298-314. In the mouse bioassay for Botulinum Neurotoxin, large numbers of mice are infected with the sample and observed for symptoms of infection and eventual death. Although sensitive (LOD ˜0.01 ng/mL of sample), this assay may require large animal populations and be quite time-consuming, often requiring >4 days.
Because of the high toxicity of Botulinum Neurotoxin, rapid diagnosis of the toxin is often important for enhanced survival rates. Additionally there are a limited number of laboratories capable of performing a mouse bioassay. While a variety of immunoassay and enzyme activity-based formats have been developed, the sensitivity and utility of these assays remains inferior to the mouse bioassay. In particular presently available assays may only provide a partial indication of the many factors ultimately linked to toxicity and best course of therapeutic intervention. For example, both light chain and heavy chain portions of the molecule are required for BoNT toxicity. In addition, BoNT complex proteins may play a critical role in systemic uptake. BoNT light chain may be present but not in the active form. Presently available assays may not distinguish between the presence of an active or inactive form of the light chain. In many cases, the BoNT serotype must be identified to properly administer therapeutics. For these reasons, comprehensive toxicity assay approaches are preferable and serotype-specific activity assays may be advantageous for effective diagnostics and therapeutic intervention.